1. Field of the Invention
This invention concerns a method for in vivo, ex vivo and in vitro repair, regeneration, de novo formation and remodeling of diseased and normal mesenchymal or mesenchymally derived cells, cartilage, collagen and bone. In particular, this invention concerns in vivo, ex vivo and in vitro regeneration of articular cartilage and collagen and bone remodeling by intermittently applied hydrostatic pressure. The method involves the application of external interval loading consisting of repeated periods of applied hydrostatic pressure followed and interrupted by periods of recovery. The method specifically concerns application of the intermittent hydrostatic pressure at levels 0.5-30 MPa for an interval of 1-8 hours followed by a recovery period up to about 16-23 hours, said pressure applied to cartilage and bone cells in vitro, explants of cartilage and bone graft ex vivo and in vivo to cartilage that remains intact within the joint space of diarthrotic joints, or in vivo to the bone. The interval loading results in significant increase in expression of proteins providing the unique phenotypic properties of cartilage and bone and in the selective inhibition of matrix degrading enzymes, pro-inflammatory cytokines and chemokines that attract inflammatory cells into the joint cavity and in selective decrease of gene expression of growth factors that are inhibitory to extracellular matrix repair and regeneration.
The invention also concerns methods for treatment of articular cartilage and collagen regeneration, restoration and transplantation.
2. Background and Related Disclosures
Arthritic diseases, particularly osteoarthritis, affect more people than any other ailment. Osteoarthritis involves loss of function of cartilage which undergoes a slow progressive degeneration in many joints. Although osteoarthritis is considered a non-inflammatory disease a certain degree of inflammation occurs. Osteoarthritis is distinguished from the rheumatoid arthritis which is a chronic inflammatory joint disease.
Osteoarthritis affects most people in late middle age. Osteoarthritic related conditions decrease personal productivity and quality of life and in an aging society, increase the morbidity and mortality for men and women by increasing the incidence of other chronic conditions, such as, for example, osteoporosis. Currently, the only successful treatment for end stage joint disease requires major surgery involving total joint replacement which is not without associated complications such as infection, aseptic loosening, and pain. These complications can then lead to the necessity for revision arthroplasty. Reversal of early onset osteoarthritis by novel surgical techniques that would abrogate the necessity for joint replacement is just now being tried in experimental stages.
Intra-articular surgical approaches are being developed that entail transfer of cartilage cells from healthy regions of the joint to diseased surfaces in order to restore joint function. In this context, cartilage cells or small regions of cartilage are placed in partial or full-thickness defects within the joint surface using an open surgical procedure. The cell construct is then held in place by periosteal tissue that is sutured in place. However, implanting cells or resurfacing with autogenous or allograft cartilage in the absence of an organized extracellular matrix does not support normal weight bearing. In many cases, these grafts quickly become fibrillated and degrade. In an alternative procedure, mosaicplasty involves moving multiple small grafts of cartilage from one area of the joint surface to another to facilitate a return to weight bearing. With any type of cartilage exchange, efficacy of repair will be greatly facilitated following restoration of an extra-cellular matrix structure of normal cartilage prior to use.
Cartilage, collagen and bone diseases, therefore, present a major medical problem, particularly with an increasing aging population which is more prone to osteoarthritis and other joint regenerative diseases, and it would thus be important to have available a means for regeneration of articulate cartilage and collagen and bone remodeling.
Articular cartilage covers the ends of long bones and is load-bearing tissue that distributes forces across joint surfaces protects the more rigid underlying bone and provides smooth articulation and bending of the joints during normal activities of daily living.
Attempts are continuously made to regenerate articular cartilage. U.S. Pat. No. 6,080,194, for example describes a collagen template formed by combining a porous collagen sponge with a collagen membrane. U.S. Pat. No. 5,786,217 describes methods and compositions for the repair of articular cartilage defects. U.S. Pat. No. 5,206,023 discloses methods and compositions for treatment and repair of defects or lesions of the cartilage. U.S. Pat. No. 5,041,138 concerns neomorphogenesis of cartilage in vivo from cell culture for the growth and implantation of cartilaginous structures. However, none of these patents disclose a method which would regenerate the diseased cartilage to a functional state and such method is still lacking.
Clinical experience in humans and experimental studies with animal models confirm that mechanical loads provide an essential stimulus for maintenance of normal articular cartilage homeostasis (Proc. Soc. Exp. Biol. Med., 190:275 (1989)).
Alterations in joint loading due to immobilization (Clin. Orthop. Rel. Res., 219:28 (1987)) ligamentous laxity (Ibid, 213:69 (1986)), excessive impact (J. Biomechanics, 6:51 (1973)) or increased subchondral bone stiffness (J. Biomechanics, 28:357 (1995)) result in pathological changes in cartilage characteristic of osteoarthritis.
The ability of cartilage to change shape rapidly and reversibly is attributable to a resilient and elastic matrix with a high content of highly soluble proteoglycans which are entrapped in collagen, an insoluble fiber network. Proteoglycans, collagen and other molecules present in the cartilage tissue are produced by mesenchymally-derived cartilage cells, the chondrocytes.
In vitro studies confirm that the cartilage cells, the articular chondrocytes, respond to specific loading conditions through an anabolic or catabolic reaction that is attributable to the stress and strain imparted to the cell by the physical stimulus (Biochem. Biophys. Res. Commun., 240:216 (1997); Spine, 22:1085 (1997) and J. Orthop. Res., 15:189 (1997)).
Recognition of the role that mechanical loading plays in the regulation of articular chondrocyte metabolism has been delineated in part by mathematical analysis of the distribution of forces across joint surfaces (J. Biomech., 22:853 (1989)).
Biomechanical analyses described in J. Exp. Physiol., 81:535 (1996) confirm that chondrocytes in the cartilage of a diarthrotic joint experience levels of hydrostatic pressure in the order of 7 to 10 MPa that result from normal activities of daily living. Studies examining the influence of mechanical forces on tissue differentiation revealed that increased cartilage thickness occurs in regions of the diarthrotic joint exposed to high intermittent compressive hydrostatic stress. Thinner cartilage coincides with regions experiencing decreased hydrostatic pressure and having tensile forces arising tangential to the joint surface (Bone, 11:127 (1990)).
Experimental studies described in J. Orthop. Res., 9:1-10 (1991) confirmed that hydrostatic pressure influences articular cartilage matrix metabolism when applied in vitro and established that hydrostatic pressure at levels of 5-15 MPa modulates 35SO4 and 3H-proline incorporation rates into adult bovine articular cartilage in vitro.
Organ culture experiments described in J. Biol. Chem., 262:15490 (1987) demonstrated that sites of proteoglycan production coincide with regions of pure hydrostatic pressure. Physiological levels of hydrostatic pressure enhance mRNA signal levels for aggrecan and type II collagen when measured immediately after loading as described in J. Orthop. Res., 14:53 (1996). In a study of load controlled compression of aggrecan mRNA expression in bovine cartilage explants a transient up-regulation was observed after 1 hour of loading.
While the above research describes and recognizes the importance of the hydrostatic pressure on normal function of cartilage and type II collagen, such knowledge was nevertheless impossible to apply clinically because the continuous application of the hydrostatic pressure leads to exhaustion of the cartilage metabolic potential and its damage while the brief ( less than 1 hour) of loading with hydrostatic pressure leads to varied cellular response which disturbs the chondrocyte metabolism and homeostasis. For example, a short period of hydrostatic pressure loading results in increased expression of type II collagen mRNA while the continuously applied load does not maintain such increased expression. On the other hand, the aggrecan signal expression continued to increase throughout the duration of the load. Clearly, these results disturb the cellular equilibrium between aggrecan and type II collagen. Cartilage cells respond to multiple stimuli in unpredictable ways and variability of the response depends on time, magnitude and frequency of loading. Clearly, this unpredictability prevents using the continuous long or short periods of indiscriminate hydrostatic pressure loading for treatment of osteoarthritis or regeneration of damaged cartilage (J. Rehab. Res. Dev., 37:153-161 (2000)).
In view of the severity and disabling effect of osteoarthritis and other cartilage, collagen or bone diseases, it would be important to provide a method which would permit a cartilage or collagen regeneration and bone remodeling.
Until recently, it was believed that articular cartilage can no longer repair itself once the arrangement of the supporting fibers has been disrupted. (Articular Cartilage and Osteoarthritis, Workshop Conference Hoechst and Werk, Kalle-Albert, Wiesbaden May 12-16, 1991, Eds. Kuettner et al., Rosen Press, New York.
It has now been found that such regeneration is possible with a specific regimen of intermittently applied hydrostatic pressure to mesenchymal or mesenchymally-derived cells, such as fibroblasts, fibrochondrocytes or chondrocytes and it is, therefore, a primary objective of the current invention to provide a method for treatment of osteoarthritis and other cartilage and collagen diseases by stimulating their regeneration, de novo formation, and bone remodeling, said method providing a defined mechanical loading environment which regenerates and repairs adult cartilage and bone cells.
All patents, patent applications and publications cited herein are hereby incorporated by reference.
One aspect of the current invention is a method for repair, regeneration and de novo formation of cartilage, replenishment of chondrocytes, or deposition of type II collagen and stimulation and bone remodeling.
Another aspect of the current invention is a method for treatment of diseases of cartilage, collagen or bone by stimulating the cartilage and collagen regeneration and bone remodeling using an interval loading regimen consisting of repeated periods of intermittently applied hydrostatic pressure within specified loading interval followed by a recovery period.
Still another aspect of the current invention is an in vivo, ex vivo or in vitro method for repair, regeneration and de novo formation of cartilage wherein the regeneration is achieved by applying an interval loading regimen consisting of repeated periods of applied hydrostatic pressure followed by periods of recovery to in situ or ex situ cartilage or cartilage cells or to cartilage or cartilage cells in vitro.
Still yet another aspect of the current invention is an in vivo or ex vivo cartilage repair, regeneration and de novo formation method which involves applying hydrostatic pressure to the cartilage tissue in need of regeneration in situ or to cartilage removed from in situ or chondrocytes, fibroblast or fibrochondrocytes adhered to a matrix and subjected to the regimen comprising applying intermittently the hydrostatic pressure for about 1-8 hours followed by about 16-23 hours of recovery period.
Still yet another aspect of the current invention is an in vivo collagen restoration method which involves applying hydrostatic pressure to the cartilage, cartilage cells, other mesenchymally-derived cells or collagen tissue in need of repair and regeneration.
Still yet another aspect of the current invention is an in vivo bone restoration method which involves applying hydrostatic pressure to the bone site in need of regeneration of cartilage as a progenitor tissue or direct mechanical stimulus using a regimen comprising applying, intermittently, the hydrostatic pressure to the bone osteoblast cells for about 1-8 hours, followed by recovery period of about 16-23 hours.
Still yet another aspect of the current invention is a diseased joint regeneration method wherein cartilage grafts that are submitted to the intermittent hydrostatic pressure loading and restored to a healthy load-bearing matrix are placed in a diseased joint to restore its normal function.
Still yet another aspect of the current invention is a regenerated functional healthy load bearing cartilage from the diseased cartilage wherein said diseased cartilage is subjected to intermittent hydrostatic pressure followed by periods of recovery until the normal mechanical and biochemical properties are restored.
Still yet another aspect of the current invention is a method for detection of functionality of the cartilage by determining relative levels of, or levels of expression of, cartilage or bone degradative enzymes, cytokines and their inhibitors or growth promoting substances, growth factors and hormones, following subjecting the diseased cartilage or bone to intermittent hydrostatic pressure followed by periods of recovery.